The present invention relates to a method of enhancing inorganic carbon fixation by photosynthetic organisms, nucleic acid molecules for effecting the method, and transformed plants characterized by enhanced inorganic carbon fixation.
Photosynthesis is a process executed by photosynthetic organisms by which, inorganic carbon (Ci), such as CO.sub.2 and HCO.sub.3.sup.-, is incorporated into organic compounds using the energy of photon radiation. Photosynthetic organisms, such as, soil grown and aquatic plants and cyanobacteria (blue-green algae), depend on the organic compounds produced via photosynthesis for sustenance and growth.
The rate of photosynthesis is determined by several parameters which include but are not limited to, CO.sub.2 concentration, O.sub.2 concentration, temperature, light intensity, and the water balance in the case of soil grown plants.
Of the above mentioned parameters photosynthesis is largely influenced by the rate with which the carboxylating enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase, (rubisco) can fix available CO.sub.2. The rate of CO.sub.2 fixation depends on the concentration of CO.sub.2 and to a lesser extent on the concentration of O.sub.2 which are available to rubisco, since CO.sub.2 fixation competes with photorespiration, which is the process of O.sub.2 fixation by rubisco. Furthermore, when photosynthesis is rate-limited by the supply of CO.sub.2, the imbalance between the light energy input and its dissipation via CO.sub.2 fixation, leads to photodynamic damage to the photosynthetic reaction centers (photoinhibition).
Since rubisco posseses a very slow turnover rate, in order to meet the energy requirements of photosynthetic organisms it needs to be present in abundance within the photosynthetic cells thereof (approximately 50% of the soluble protein). The abundance of rubisco leads to deleterious effects to the energetic balance of photosynthetic cells since most available resources of these cells must be allocated to the production of rubisco.
To overcome the problems associated with inefficient CO.sub.2 fixation at low CO.sub.2 concentrations, many photosynthetic organisms have evolved various mechanisms for concentrating the inorganic carbon exposed to rubisco. Such mechanisms are found in certain higher plants (belonging to the C4 group, including maize and sorghum) and in aquatic photosynthetic organisms. In C4 plants, differential expression and tight regulation of several genes enables cooperation between the mesophyll and bundle sheath cells leading to elevated CO.sub.2 concentration in the latter. An initial carboxylation reaction, where HCO.sub.3.sup.- serves as the substrate, occurs in the mesophyll cells. The product is then transferred to the bundle sheath cells where it is decarboxylated (releasing the CO.sub.2 fixed in the mesophyll cells) in close proximity to rubisco (confined to these cells). C3 plants, to which most of the crop plants belong, are unable to concentrate CO.sub.2 at the site of rubisco and therefore grow poorly (compared to C4 plants) under water-limiting conditions. Due to the large number of genes involved, and to the complexity of their tight, spatial regulation, in the operation of the C4 mechanism, the introduction of the whole C4 CO.sub.2 concentrating mechanism to C3 plants is presently impossible.
Many aquatic photosynthetic microorganisms possess inducible mechanisms that concentrate CO.sub.2 at the carboxylation site, compensating for the relatively low affinity of rubisco for its substrate, thus allowing acclimation to a wide range of CO.sub.2 concentrations [25]. The presence of membrane-located mechanisms for inorganic carbon (Ci) transport are central to these concentrating mechanisms.
Photosynthetic microorganisms including cyanobacteria are also capable of acclimating to a wide range of CO.sub.2 concentrations. The process of acclimation is mediated via changes, effected at various cellular levels, which include modulation of gene expression involved in the operation of the CO.sub.2 concentrating mechanism (CCM) [1-5]. This mechanism enables these photosynthetic microorganisms to raise the CO.sub.2 level at the carboxylation site thus overcoming the large (5 to 20-fold) difference between the K.sub.m (CO.sub.2) of rubisco and the concentration of dissolved CO.sub.2 when at equilibrium with the surrounding atmosphere. In cyanobacteria, the components of the CCM include energy-dependent HCO.sub.3.sup.- transport, CO.sub.2 conversion to HCO.sub.3.sup.- [3] and highly organized structures, termed carboxysomes, where carbonic anhydrase catalyzes the formation of CO.sub.2 from HCO.sub.3.sup.- in close proximity to rubisco [1-3]. The activity of the CCM increases dramatically following transfer from high to low CO.sub.2 concentrations mainly due to changes in the Ci transport capabilities and an increase in the number of carboxysomes [3, 6, 7]. Some of the genes involved in the operation of the CCM were identified with the aid of high-CO.sub.2 -requiring mutants but there is little information on those directly involved in HCO.sub.3.sup.- uptake [3, 4, 8].
The ability to concentrate CO.sub.2 provides distinct advantages to photosynthetic organisms. The photosynthetic rate of such CO.sub.2 concentrating organisms is not severely affected by lower CO.sub.2 concentrations and as a result, the growth and productivity of such organisms are not severely limited by the environmental concentration of CO.sub.2, and by water, in the case of soil grown plants.
Therefore, it is highly desirable to enhance CO.sub.2 fixation in non CO.sub.2 concentrating photosynthetic organisms, such as C3 plants, since in all probability, such an enhancement would directly result in improved growth and productivity.
There are two possible biotechnological approaches with which an enhanced rate of CO.sub.2 fixation can be achieved.
One such approach involves the manipulation of rubisco by site directed mutagenesis in order to raise both its affinity and specificity to CO.sub.2 (compared with O.sub.2) and to further enhance its turnover rate. Although numerous studies were conducted in order to isolate a rubisco mutant which posseses the above mentioned improvements at present no such rubisco mutants have been isolated.
Another approach for enhancing CO.sub.2 fixation involves raising the concentration of CO.sub.2 outside the cells of higher plants. In higher plants the concentration of CO.sub.2 in the air spaces within the leaves is determined by the diffusional flux of CO.sub.2 through the stomata from the surrounding air via the unstirred layer around the leaves. The stomatal conductance for CO.sub.2 is largely determined by the water balance of the plants. Modulation of stomatal opening by water availability is exercised by plants in order to combat water stress. Stomatal closure, in order to minimize water loss under conditions of water stress, generates a significant resistance to CO.sub.2 diffusion leading to a sharp decline in CO.sub.2 fixation. Although raising the concentration of CO.sub.2 in a closed environment, such as a greenhouse, is commonly practiced in order to raise the diffusional flux of CO.sub.2 and as such, raise plant productivity, such a practice however, is not applicable to outdoor grown plants.
Increasing stomatal conductance can theoretically serve to raise plant productivity, but at present, viable mechanisms for enhancing the stomatal CO.sub.2 conductivity have not been proposed.
Thus, at present, both of the above mentioned approaches are theoretical and as such cannot be applied to render photosynthetic organisms, such as C3 plants, more efficient at fixing CO.sub.2.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of enhancing the CO.sub.2 fixation in photosynthetic organisms, such as higher plants, especially C3 plants.